Taxonomy and chemically semi-defined media for the analysis of the tacrolimus producer ‘Streptomyces tsukubaensis’

Abstract

'Streptomyces tsukubaensis' was the first tacrolimus producer strain identified. Although it has been included in the Streptomyces genus, its taxonomic position has not been rigorously determined. By using a polyphasic approach, we have established that the tacrolimus producer strain 'S. tsukubaensis' NRRL 18488 represents a unique species in the Streptomyces genus, which is phylogenetically distant from other subsequently described producers. This fact means a horizontal transference of the tacrolimus-producing gene cluster. Physiology, nutrient requirement, and molecular genetics analyses of tacrolimus biosynthesis in 'S. tsukubaensis' necessitate chemically defined or semi-defined media, which work as a jigsaw puzzle and allow for pieces (nutrients) exchange. To date, studies related to 'S. tsukubaensis' have been mainly focused in the improvement of tacrolimus production using complex industrial fermentation media, which difficulty allows testing of tacrolimus overproduction enhancers or inhibitors because of the presence of non-defined substances. In the present work, two semi-defined media were developed in order to study the main factors involved in tacrolimus production in 'S. tsukubaensis'.

Phage P1-Derived Artificial Chromosomes Facilitate Heterologous Expression of the FK506 Gene Cluster

“…Upon cultivation in MGm 2.5 medium [20], production of FK506 was detected in all strains containing PAC20N using HPLC-UV and HPLC-MS in comparison to an authentic FK506 standard (Inresa Pharma, Bartenheim, France). Figure 3 shows representative chromatograms from strain M1146/PAC20N.…”

“…Streptomyces tsukubaensis NRRL 18488 was obtained from the US Department of Agriculture strain collection and grown on ISP4 plates [20]. Mycelia were prepared for PAC library construction by growing S. tsukubaensis in YEME medium (100 ml) inoculated with mycelia from a TSB starter culture (10 ml, [37]).…”

“…The mycelia were subsequently homogenized [52] and stored at −80°C to serve as stocks for inoculation of production medium. To measure heterologous production of FK506, each strain was grown in 24-well microtiter plates (28°, 300 RPM, [4], [52]) for 6 days in MGm 2.5 medium ([20] with minor modification). Each well of the 24-well microtiter plate contained 3 ml of MGm 2.5 media and was inoculated with 75 µl of homogenized mycelia (3 wells per culture replicate).…”

“…FK506 is a high value calcineurin inhibiting immunosuppressant [19], marketed under the trade name Prograf with annual sales of approximately $2 billion (evaluatepharma.com, [20]). It is now used following bone marrow, kidney and liver transplants [21], [22], [23], is effective at treating skin inflammation and eczema [24], [25] and is gaining additional attention for its potential in treating cancer [26] and neurological conditions [27].…”

“…Genomic information for FK506 producing Streptomyces has only recently emerged [36], and targeted genetic manipulations in some producing strains are hindered by slow growth or difficulties with introduction of exogenous DNA. In addition, FK506 production is sensitive to a number of culture conditions including pH and the concentrations of lysine, nitrogen, and phosphate [20].…”